The effects of a diet with high fat content from lard on the health and adipose-markers’ mRNA expression in mice

Abstract

Pork is one type of the most frequently consumed meat with about 30% globally. Thus, the questions regarding to the health effects of diet with high fat content from lard are raised. Here, we developed a model of mice fed with high fat (HF) from lard to investigate and have more insights on the effects of long-time feeding with HF on health. The results showed that 66 days on HF induced a significant gain in the body weight of mice, and this weight gain was associated to the deposits in the white fat, but not brown fat. The glucose tolerance, not insulin resistance, in mice was decreased by the HF diet, and this was accompanied with significantly higher blood levels of total cholesterol and triglycerides. Furthermore, the weight gains in mice fed with HF seemed to link to increased mRNA levels of adipose biomarkers in lipogenesis, including Acly and Acaca genes, in white fat tissues. Thus, our study shows that a diet with high fat from lard induced the increase in body weight, white fat depots’ expansion, disruption of glucose tolerance, blood dyslipidemia, and seemed to start affecting the mRNA expression of some adipose biomarkers in a murine model.

Introduction

A high fat (HF) is described as a diet containing lipids that contribute to over 30% of the total calorie intake, and it may be up to 50%, according to some current research. ¹ US data for the period from 1908 to 1985 showed that the proportion of total energy intake from carbohydrates fell from 57% to 46%, while the total calorie intake from fats grew from 32% to 43%. ² The trends for carbohydrate and fat consumption were still maintained in the following years, which reached 35% and 45%, respectively. ² Various physical health consequences are linked to HF such as obesity, diabetes, intestinal diseases, autoimmune disorders, and several types of cancer.^3,4 An HF-induced imbalance between energy intake and expenditure causes a redundant accumulation of fat in white adipose tissue (WAT), which may lead to obesity and diabetes. ⁵ The excessive intake of saturated fats, including a minute amount of omega-3 polyunsaturated fatty acids (PUFAs) and a considerable amount of omega-6 PUFAs is associated with the abnormality of metabolism. ² HF could also induce abnormal liver tissue, diversity loss of intestinal microbiota and inhibit intestinal development. ⁶ In terms of autoimmune disorders such as systemic lupus erythematosus, the association between diet and their risk was suggested around 50 years ago. ⁷ Moreover, HF aggravates prostate cancer, pancreatic cancer and colitis-related carcinogenesis.^8–10

The burden of HF is undeniably immense in mental health, economy and environment. HF-induced obesity also has a notable association with attention deficit hyperactivity disorder, bipolar disorder, depression and substance abuse. ¹¹ Furthermore, weight stigma was negatively related to self-esteem and positively related to body image dissatisfaction, anxiety and eating disturbances. ¹² Obesity-induced stress caused by HF accelerates hair thinning by targeting hair follicle stem cells. ¹³ Obesity also aggravates sebaceous glands causing acne, increased skin infection incidence and premature hair graying. ¹⁴ The medical costs for treating such diseases in the USA are expected to reach $66 billion in 2030. ³ Obese patients perform less effectively at work due to their low life expectancy and physical limitations. ³ Productivity loss caused by hospitalization and low life expectancy was estimated to be $74 million and $444 million in the USA, respectively. ³ The consequences of HF disease have also been proven for the environment. A study has shown diets in the Xinjiang Autonomous Region, which includes high carbohydrates and fats intake were associated with high ecological footprint, high water footprint and high carbon footprint. ¹⁵

Pork is one of the most frequently consumed meats, with about 30% globally. In lard, we can find about 46% monounsaturated (MUFAs), about 37% saturated (sFA), and about 17% polyunsaturated (PUFAs). Lard contains extremely high levels of oleic acid (44%), palmitic acid (27%) and stericand oleic acid (11%). ¹⁶ Because of the composition, lard has been recommended and a commonly used as fat source in metabolic studies on mice. These diets have been particularly effective in mimicking the metabolic effects of the human diet due to the significant proportion of lard consumption in human diets in the world. Notably, Vietnam ranked fifth among the top 10 largest pork consumers in 2021 of the world. ¹⁷ This issue raises the question of how over-eating fat from pork lard affects health and how we can control this problem in the countries that consume the most pork particularly and globally. To set the light on the effect of HF from pork fat on health, it is necessary to develop animal models to sufficiently mimic all characteristics of human disease. Among prevalent animal models, mice are widely applied to explore HF impacts due to reasonable prices and faster growth with less feed consumption. ¹⁸ Therefore, murine HF has been used to model type-2 diabetes mellitus, wound healing, Alzheimer's, aging, dysbiosis and prostate disease.^18–22 To investigate and have more insights on the effects of longtime-feeding with HF on the health, we have developed a model of mice fed with HF from pork lard in Vietnam. The effect of the HF has been comprehensively evaluated through mouse and fat tissues weight, glucose tolerance and insulin resistance, blood lipids and liver enzymes, and adipose biomarkers of lipogenesis.

Methods

Animal models

The 6-week-old male Swiss mice or other suitable strains were purchased from the National Institute of Hygiene and Epidemiology and raised at the animal laboratory at the Center for Biomedicine and Community Health, VNU-International School. The procedure for building animal models has been described in detail in our previous studies.^23,24 Six-week-old mice were kept in a cage and adaptably housed at room temperature and fed ad libitum with a standard diet for acclimatization until starting of the experiment at the 8 weeks of age. Our study adhered to institutional guidelines concerning the care and utilization of laboratory animals.

Then, mice were guaranteed to eat 6 g of food/mouse/day during the protocol. Mice were divided into two groups through simple randomization method by lottery. Each group has 18–19 mice and 3–4 mice were raised per cage. One group was fed the standard diet (STD) with 5.53% kcal of fat, and the other was provided a HF with 60% kcal from fat. In the STD, 5.53% kcal of fat was included totally in 6 g of standard rodent pellets/mouse/day purchased from the National Institute of Hygiene and Epidemiology. On the other hand, the fat in the HF is obtained from pork hard fat. It was washed, boiled, chopped, and mixed with standard rodent pellets. Specifically, HF group was fed 2.55 g of standard rodent pellets and 3.45 g of lard/mouse/day to ensure a ratio of 60% kcal of fat. Throughout the protocol, mice were always housed at 25 °C with a 12-h light/dark cycle. The mouse cage has been cleaned daily by replacing the coop liner with sterilized and dried sawdust. The mouse's weight was monitored using the SF400D scale (minimum division 0.01 g) every two days. When any mouse with abnormal signs was detected, it would be separated into new cages to avoid affecting to other animals.

The experiment ended on the 66th day of STD and HF feeding. The mice were sacrificed to collect the tissues and blood for further analyses. The inguinal (ING WAT) and epididymal (eWAT) white fat tissues, and interscapular brown adipose tissue (iBAT) were collected, kept in 1.5 ml Eppendorf tubes and weighed, then were instantly frozen in liquid nitrogen and stored at −80 °C until total RNA isolation.

Glucose tolerance test (GTT) and insulin tolerance tests (ITT)

GTT and ITT were performed after the mice had fasted for 12 h, as described. ²⁴ For GTT, we diluted 30% glucose solution to 20% glucose solution with distilled water, then intraperitoneal injection of 20% glucose solution was used following the 2 g/kg body weight ratio. After GTT 2 days, mice were conducted ITT, the 100 IU/ml insulin solution was diluted 1000 times to form a 0.1 IU/ml insulin solution, then mice were injected with intraperitoneal insulin at 0.75 IU/kg of body weight. Blood glucose levels were measured using an On Call Plus II Blood Glucose Meter (ACON Laboratories, Inc., USA) with five-time points: before injection, 15 min, 30 min, 90 min, and 120 min after injection.

Measurement of blood lipids and liver enzymes

Fasting blood samples from the heart of each mouse were collected. We cut the artery of the mouse's heart and used a 5 ml syringe to suck the blood and put it in a Heparin tube. Plasma was obtained by centrifuging a blood sample immediately after collection. It was then stored at −80 °C until blood lipids and liver enzymes were tested. Triglyceride (TRI), Total Cholesterol (CHO), High-Density Lipoprotein-cholesterol (HDL), and Low-Density Lipoprotein-cholesterol (LDL) were determined to assess. Liver function has been evaluated through indicators such as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT). All the above analyses were performed using an automated blood analyzer (Type Architect C8000; Abbott Ltd, USA) at the Genome Sciences Technology And Services Company Limited, Hanoi, Vietnam.

RNA isolation and QRT-PCR

Total RNA was isolated from frozen fat tissues by the QIAamp viral RNA mini Kit (Qiagen, 52906). The total RNA's absorbance qualities and purification levels were assessed using a NanoDrop spectrophotometer. The ratio of absorbance at 260 nm versus 280 nm (260/280), and 260 nm versus 230 nm (260/230), as well as the total RNA concentration (ng/μl) were recorded. Total RNA was used to perform Quantitative reverse transcription-PCR (qRT-PCR). We used the TaqMan probe for Acly and Acaca genes available from previous studies upon request. Acly and Acaca can be considered as biomarkers of adipose tissue because it directly involved in fatty acid synthesis, an important process in adipose tissue.^23,25,26 All mRNA expression data has been normalized by using the cyclophilin as the reference gene.

Data analysis

The data was analyzed by STATA 14 (StataCorp LLC, Texas, USA) and GraphPad Prism 8.0. It was expressed as mean ± SEM. Student's t-test and two-way ANOVA tests were used for single comparisons and multiple comparisons, respectively. Significance was accepted at p < 0.05 and not significant (n.s.) as p ≥ 0.05.

Results

Effect of the high fat diet on mouse body weight

Eight-week-old mice were fed with HF or STD in 66 days to check the effect of HF on the health and mRNA expression of some adipose biomarkers of lipogenesis. The amount of food consumed by the two groups did not differ during the experiment. The results showed a significant increase in mouse body weight in the HF group but almost no change in the STD group. In particular, the mice's body weight increased rapidly in the first 30 days in response to HF, then continued to increase slightly until the 66th day. On the opposite side, the body weight was slightly decreased but not statistically in the group of mice fed with STD. When comparing the body weight of mice in the two groups (Figure 1(a)), our data indicated that body weight did not significantly differ between the two groups at the start of the experiment (Day 0). Thereafter, body weight was significantly higher at HF than STD on test days 10, 30, and 66 at 6.45(g), 9.97(g), and 12.79(g) body weight, respectively. Obviously, we calculated the changes in body weight of mice by the diets at the experimental time points versus the day of starting (Day 0) (Figure 1(b)), the data clearly shows that the mice body weight was significantly changed by HF, up to the peak of 9.27 (g) at the day 30, then still high and reached 10.23 (g) at the end of the experiment, but the STD did not seem to make the significantly changes in animal bodyweight. Thus, the current data show that after 66 days fed with HF in our laboratory condition, mice's weight was significantly elevated compared to those fed with STD, and also versus the beginning of experiment.

Figure 1.

Effect of the high fat diet on the body weight of mice. Adult mice at eight weeks of age were fed with HF or STD for 66 days. A. Body weight of mice was measured every two days. B. The mouse body weight was compared between HF and STD groups at the selective time points (0, 10, 20, 20, 40, 50, and 66 days from starting day of the experiment – Day 0). C. The changes in mouse body weight every two days compared to Day 0 in each mouse group. D. The heat map representing the changes in mouse body weight in HF and STD groups at each time point during the course of experiment. Student's t-test was performed as the statistical test. (n = 18–19 mice per group).

Effect of the high fat diet on the weight of fat tissues

One of the most lipid-accumulative tissues in animals is white adipose tissue. We have weighed the fat tissues of mice fed by STD and HF (Figure 2). The current data showed that the HF affected the weight accumulation of the WAT, but not BAT. Particularly, the ING WAT and eWAT were significantly increased by HF compared to STD by about 15% and 30%, respectively. The increase in ING WAT and eWAT seems to reflect the increase in the body weight of animals by HF. On the other hand, the data clearly indicate that the HF did not significantly change the weight of interscapular brown adipose tissue (iBAT). Therefore, this result proved that the HF made the accumulation in the body weight when a significant increase in white adipose tissue appears.

Figure 2.

Effect of the high fat diet on the weight of fat tissues. The mice were sacrificed at the end of the experiment – day 66. The ING, eWAT, and iBAT were collected from every mouse in both STD and HF groups, then the tissues were quickly weighed to compare the effects of diet. Student's t-test was performed as the statistical test. (n = 18–19 mice per group).

Effect of high fat diet on glucose tolerance and insulin resistance

One of the health effects of the longtime eating of high fat-contained-food is the induction of the disruption of the lipid and glucose metabolism with the initial signs of dysfunction of glucose tolerance and insulin resistance. We have conducted GTT and ITT for the two groups of animals fed with STD or HF for 66 days (Figure 3). As expected, the HF significantly affected the glucose tolerance of the experimental mice, that is the HF-fed-mice had the higher blood glucose levels in 15, 30, and 90 min compared to the STD group after GTT test. After that, blood glucose levels cannot return to normal levels as seen in STDs at the end of the GTT test (120 min) (Figure 3(a) and (b)). But unexpectedly, we did not see any difference between HD and STD in the glucose levels in the mouse blood after insulin injection during the ITT test (Figure 3(c) and (d)), as we found in previous work when we fed B6 mice with a HF diet for 154 days (22 weeks). ²⁴ In that report, 154 days on HF increased the blood glucose levels in the ITT test compared to the STD-fed-mice group. ²⁴ So this result means that mice had reduced glucose tolerance for 66 days in HF but were not induced with insulin resistance.

Figure 3.

Effect of the high fat diet on glucose and insulin tolerance. Before finishing experiments 4 and 2 days, we have done the GTT and ITT. A. The glucose levels in mouse blood in two diet groups were measured at 0, 15, 30, 90, and 120 min after glucose injection. B. The comparison of blood glucose levels in HF versus STD group at the indicated time points in GTT. C. The blood glucose levels of mice in two diet groups were measured at 0, 15, 30, 90, and 120 min after insulin injection in ITT. D. Blood glucose levels in HF-fed mice were compared to those of the STD group at the indicated time points in ITT. Student's t-test was performed as the statistical test. (n = 18–19 mice per group).

Effects of the high fat diet on the blood lipids and liver enzymes

Furthermore, we looked more closely at the effects of high fat feeding on the lipid metabolism of animals by checking profiles of the lipids and liver enzymes in the blood of experimental mice in two groups (Figure 4). In 66 days of eating the food with high content of fat, the levels of blood lipids were changed selectively (Figure 4(a)). Specifically, we saw that CHO and TRI levels were significantly elevated by the high fat feeding, which was clearly higher than those in the standard diet group (P < 0.01). However, the 66 days in HF only slightly increased the blood levels of HDL and LDL, and this increase was not statistical significance. Furthermore, the plasma levels of GOT and GPT were not different between mice in HF versus STD groups (Figure 4(b)).

Figure 4.

Effects of the high fat diet on the blood lipids and on liver enzymes. The mouse blood was collected from each mouse in both groups at the end of the experiment to check plasma lipids and liver enzyme levels. A. The levels of 4 types of most important blood lipids, including CHO and TRI, HDL and LDL in mice fed with HF versus STD. B. The levels of plasma liver enzymes – GOT and GPT in HF versus STD group. Student's t-test was performed as the statistical test. (n = 18–19 mice per group).

Effects of the high fat diet on adipose biomarkers of lipogenesis

In this study, we deeply investigated if the longtime-eating-high fat affected the mRNA expression of the adipose markers related to the lipid metabolism, and we selected to check the mRNA levels of two important lipogenic genes including ATP Citrate Lyase (Acly) and Acetyl-CoA Carboxylase Alpha (Acaca) in white and brown fat tissues of the mice in two groups. We found that the effects of HF on mRNA levels of Acly and Acaca genes in fat tissues (Figure 5) seems to reflect those effects on the changes on the fat tissues and animals’ body weight (Figure 2). That is, except the mRNA level of Acaca in eWAT, the high fat feeding significantly increased the mRNA levels of the Acly and Acaca genes in the white adipose tissues including ING WAT (Figure 5(b)) and eWAT (Figure 5(c)) (p < 0.05). While the p-value indicates no statistical significance, the mean differences observed suggest a potentially meaningful biological effects in mRNA levels in the classical brown fat tissues (iBAT) (n.s.) (Figure 5(a)). This result suggests that the longtime on HF effects not only on the changes in body weight, white fat depots’ expansion, the ability of glucose tolerance, the blood lipids’ levels, but also affect the mRNA expression of the adipose biomarkers.

Figure 5.

Effects of the high fat diet on adipose biomarkers of lipogenesis. The mRNA levels of selective adipose biomarkers functioning on the lipogenesis were quantitated by RT-PCR in the fat tissues collected from mice at the end of experiment. A. mRNA levels of Acly and Acaca in iBAT of HF versus STD group. B. mRNA levels of Acly and Acaca in the ING of HF versus STD group. And C. mRNA levels of Acly and Acaca in eWAT of HF versus STD group. Student's t-test was performed as the statistical test. (n = 7 mice per group).

Discussion

Nowadays, the prevalence of metabolic disorders is increasing day by day. ²⁷ Obesity has been considered a global health problem and a major risk factor for chronic diseases and cancer. ²⁸ Thus, scientists have been trying to generate mouse models to elucidate the influence of HF on mouse development, especially on the physiological mechanisms of obesity. Although this disease is related to genetic factors, it is largely caused by diet and exercise habits. ²⁸ A series of tests have consistently shown that there was a difference in weight between HF-fed mice and standard-fed mice after follow-up timelines.^29–31 The increase in body weight in mice with HF was thought to be between 10% and 20% of standard-feeding mice. However, the change in weight between each group depends on the fat composition of each diet.^32,33 The researchers also demonstrated that weight changes were associated with different strains of mice when it was fed by HF. ³³ On the contrary, some scientists have asserted that HFs do not cause weight gain if they do not exceed the threshold of total daily calories, even if those mice had up to 90% of their total calories coming from HF. ³⁴ The reason for this mechanism is due to the homeostasis and pleasure system of the brain. In our laboratory condition, we found that the 66 days fed with HF the weight of mice was significantly higher in comparison to the STD, and the weight gain of mice in HF group was reached at around 10 (g) versus the beginning of the experiment.

Based on the function, adipose tissues are classified into white and thermogenic fat tissues. The white adipose tissues (WAT) function to store energy as lipids and locates along the body especially under the skin such as the inguinal white adipose tissues (ING WAT), and around or in visceral such epididymal white adipose tissue (eWAT), perirenal white adipose tissue (prWAT), retroperitoneal white adipose tissue (rWAT), and mesenteric white adipose tissue (mWAT). ³⁵ But the thermogenic fat tissues function to burn lipids into heat, and they include interscapular brown adipose tissue (iBAT), and brite/beige adipose tissues inside the white fat deports. ³⁶ Thus, in this work we examined the effect of longtime-feeding by a HF from lards on the weight of the ING WAT, eWAT and iBAT in mice, because the ING WAT and eWAT are typical white fat tissues, and iBAT is typical thermogenic fat tissues, and we found that the HF diet significantly increased the expansion of ING WAT and eWAT, but not iBAT, this is continent with the increase in the bodyweight of mice induced by the HF (Figure 2).

Moreover, we further found that the significant increase in the body weight of mice by HF was accompanied with the dysfunction glucose tolerances, and the elevation in the weight of white fat tissues but not classical brown fat. Lipid metabolism involves the biosynthesis of lipid particles like fatty acids, cholesterol, or triglycerides as well as their degradation. HDL is known for its function in removing excess cholesterol, and LDL is related to plaque formation. CHO, HDL, and LDL are commonly utilized to assess overall lipid metabolism and cardiovascular risk, while TRI is a key indicator of lipid metabolism. Thus, these four indexes are the most important and popular indicators to predict the detrimental effects of HF on lipid metabolism and health^37,38 According to Li et al. (2020), mice feeding with HF had dramatically higher levels of serum TRI, CHO, and LDL, while HDL was substantially lower than those in the STD group. ³⁹ Consistent results from similar studies were indicated that dyslipidemia was often accompanied by HF and obesity. ⁴⁰ HF also accelerates the excessive accumulation of lipid droplets and oxidase stress by breaking the lipid synthesis and decomposition balance. We observed similar results with Li et al. (2020), in which longtime fed with HF from pork hard fat remarkably increased TRI and CHO levels, suggesting dyslipidemic induction. Moreover, the elevated TRI is associated with a higher risk of atherosclerosis, occurring when fatty deposits build up in the walls of arteries, and restrict blood stream. ⁴¹ The elevated CHO contributes to plaque formation in arteries, increasing the risk of coronary heart disease. ⁴² Thus, a long intake of saturated fats, like pork hard fat, which is one of the main worldwide food sources, may lead to a higher risk of several cardiovascular consequences and metabolic syndromes. However, we saw only slight changes in the blood levels of HDL and LDL, this may be due to the differences in experimental conditions and times of feeding HF, and origins of fat content between laboratories. Another hypothesis is that an adaptive metabolic response may occur and moderate the changes in LDL and HDL.

It has been reported that HF promotes liver dysfunction by regulating the plasma level and activity of liver enzymes. HF negatively affected liver function in C3 h mice treated with dietondiethylnitrosamine, as indicated by increased alkaline phosphatase (ALP), GPT, and GOT plasma levels and increased γ-glutamyltransferase activity. ⁴³ Matsumoto M. et al. stated that HF increased serum GPT in male A/J mice and C57BL/6J mice. ⁴⁴ Hamsters fed by HF also had significantly higher GOT and GPT plasma levels. ⁴⁵ Since GOT and GPT are present in hepatic cells, the increased plasma level of GOT and GPT is one of the earliest signals of hepatic injury. However, unexpectedly, the plasma levels of GOT and GPT were not affected by 66 days of feeding a diet with high fat content from lard in our experiment. This finding can be explain by HF can affect liver function, however this change may not occur immediately. Since the 66-day duration of HF may not be long enough, the liver is thought to be temporarily able to adapt to changes in fat metabolism without causing any significant changes in liver enzyme levels.

The changes in the expression level of lipogenesis genes are thought to be directly related to diet-induced obese mice. Acaca is a paramount gene in controlling fatty acid synthesis, while Acly has been shown to highly affect histone acetylation and adipocyte differentiation. ⁴⁶ In our HF-fed-animals, we found that the mRNA levels of Acly and Acaca genes in the white adipose tissues, including ING WAT and eWAT were elevated compared to the STD group. Voigt et al. (2013) indicated that HF could elevate the expression of both Acaca and Acly in eWAT, which was consistent with our results. ⁴⁷ Another similar study suggested that age and sex could be responsible for the difference in gene expression level of adipogenesis gene, in which Acaca expression level in HF-fed females was significantly higher than in males, thus leading to higher metabolic activity and lower risk of obesity. ⁴⁸ However, controversial results were still reported. When investigating the mRNA expression levels of several genes related to fatty acid synthesis like Acaca and Scd1 among HF mice, scientists found that the gene expression of Scd1 was even significantly higher in their offspring compared to those of STD mice while this trend was not found in Acaca. ⁴⁹ Besides, results from Jian Dong et al. (2024) indicated that both mRNA and protein expression of Acaca in hepatic cells were increased in response to the HF diet, right after 2 weeks of fat intake. The authors also reported that the lipid accumulation process, especially the plasma level of TG and CHO, and mitochondrial function were declined by inhibiting Acaca via in vitro model. Thus, Acaca was considered as a potential target for therapeutic development in obesity and non-alcoholic fatty liver disease. ⁵⁰ On the other hand, we found that there was no statistically significant difference in gene expression in iBAT, but the mean differences observed suggest a potentially meaningful biological effect. Future studies with larger sample sizes are suggested to shed more light on this finding.

Although our study has shed light demonstrate that a high-fat diet containing lard leads to an increase in body weight, expansion of white fat depots, disruption of glucose tolerance, blood dyslipidemia, and appears to initiate alterations in the mRNA expression of certain adipose biomarkers in a murine model. However, our study still has limitations that need to be considered. Firstly, we selected the number of mice without using a specific sample size calculation formula. However, we chose the sample size based on building animal models in the previous study.^23,24 Secondly, the experiment lasted for 66 days. Although this period allows for the observation of certain metabolic changes, extended studies might be required to comprehensively understand the long-term effects of dietary interventions on metabolism and health outcomes.

Conclusion

In this study, we performed a mice model to clarify the effect of a high-fat diet form lard on several indexes. The current data suggest that the 66 days exposed to a HF diet induced the increase in body weight, white fat depots’ expansion, disruption of glucose tolerance, blood dyslipidemia with the total cholesterol and triglycerides, and seemed to start affecting the mRNA expression of some adipose biomarkers in a mouse model. However, we observed no significant changes in iBAT, insulin resistance, other blood lipid indexes like HDL and LDL, and hepatic function. Future researches should deeply delve into explaining the underlying mechanisms of these above observations. Longitudinal studies with extended dietary interventions and larger sample sizes may unveil nuanced metabolic changes and shed light on the temporal progression of metabolic dysfunction. In conclusion, a long-term intake of high fat from lard had a detrimental effect on health and a potential induction of the changes in the expression levels of lipogenesis genes in mice.